Abstract Ten years ago, we found that a brief exposure to 10-50 mM ethanol prior to cardiac ischemia reduces infarct size by ~70% in a process that is dependent on activation of epsilon protein kinase C, [unreadable]PKC. In the past funding period, we found that activation of the mitochondrial enzyme, aldehyde dehydrogenase 2, ALDH2, appears to be required and sufficient for ethanol-induced cardiac protection from ischemia. The importance of mitochondrial ALDH2 in human health is also suggested by the increased propensity of 40% of East Asians that carry an inactivating mutation in the Aldh2 gene, Aldh2*2, to have a variety of chronic diseases associated with oxidative stress and the resulting accumulation of toxic aldehydes, including myocardial infarction. We plan to determine whether ethanol-induced cytoprotection requires [unreadable]PKC and ALDH2 activity, using genetically manipulated mice (AIM 1A). We will next identify the mechanisms that enable ethanol-induced entry of the cytosolic [unreadable]PKC into the mitochondria, where ALDH2 is found, (AIM 1B). We will then determine whether ALDH2 activation by ethanol and other activators reduces aldehydic adduct loads to reduce cytotoxicity (AIM 1C). We will determine whether acetaldehyde, which accumulates on ethanol treatment, contributes to ethanol-induced cardioprotection (AIM 1D) and determine whether ethanol- induced and [unreadable]PKC-mediated phosphorylation of ALDH2 protects ALDH2 activity from inactivation by long chain aldehydes and whether the effect is additive with new small molecule activators of ALDH2, called Alda (AIM 1E). In AIM 2, we will study the loss of ethanol-induced cardioprotection due to ALDH2 inhibition by nitroglycerine (NTG). We will identify small molecule that inhibit NTG-induced ALDH2 inactivation (NTG tolerance) (AIM 2A, B) and will determine the effect of NTG tolerance inhibitors on ethanol-induced [unreadable]PKC- mediated cardioprotection from acute ischemic damage ex vivo and in animal models (AIM 2C). Together, these studies will elucidate fundamental processes associated with cytoprotection in animals with wildtype and inactive (ALDH2*2) form of ALDH2 and how moderate ethanol consumption affects them. Our studies will also provide new tools and test their application as treatment for cardiac ischemia using animal models.